Tom22 is a preprotein receptor and organizer of the mitochondrial outer membrane translocase complex (TOM complex). Rat Tom22 (rTOM22) is a 142-residue protein, embedded in the outer membrane through the internal transmembrane domain (TMD) with 82 N-terminal residues in the cytosol and 41 C-terminal residues in the intermembrane space. We analyzed the signals that target rTOM22 to the mitochondrial outer membrane and assembly into the TOM complex in cultured mammalian cells. Deletions or mutations were systematically introduced into the molecule, and the intracellular localization of the mutant constructs in HeLa cells was examined by confocal microscopy and cell fractionation. Their assembly into the TOM complex was also examined using blue native gel electrophoresis. These experiments revealed three separate structural elements: a cytoplasmic 10-residue segment with an acidic α-helical structure located 30 residues upstream of the TMD (the import sequence), TMD with an appropriate hydrophobicity, and a 20-residue C-terminal segment located 22 residues downstream of the TMD (C-tail signal). The import sequence and TMD were both essential for targeting and integration into the TOM complex, whereas the C-tail signal affected the import efficiency. The import sequence combined with foreign TMD functioned as a mitochondrial targeting and anchor signal but failed to integrate the construct into the TOM complex. Thus, the mitochondrial-targeting and TOM integration signal could be discriminated. A yeast two-hybrid assay revealed that the import sequence interacted with two intramolecular elements, the TMD and C-tail signal, and that it also interacted with the import receptor Tom20. Tom22 is a preprotein receptor and organizer of the mitochondrial outer membrane translocase complex (TOM complex). Rat Tom22 (rTOM22) is a 142-residue protein, embedded in the outer membrane through the internal transmembrane domain (TMD) with 82 N-terminal residues in the cytosol and 41 C-terminal residues in the intermembrane space. We analyzed the signals that target rTOM22 to the mitochondrial outer membrane and assembly into the TOM complex in cultured mammalian cells. Deletions or mutations were systematically introduced into the molecule, and the intracellular localization of the mutant constructs in HeLa cells was examined by confocal microscopy and cell fractionation. Their assembly into the TOM complex was also examined using blue native gel electrophoresis. These experiments revealed three separate structural elements: a cytoplasmic 10-residue segment with an acidic α-helical structure located 30 residues upstream of the TMD (the import sequence), TMD with an appropriate hydrophobicity, and a 20-residue C-terminal segment located 22 residues downstream of the TMD (C-tail signal). The import sequence and TMD were both essential for targeting and integration into the TOM complex, whereas the C-tail signal affected the import efficiency. The import sequence combined with foreign TMD functioned as a mitochondrial targeting and anchor signal but failed to integrate the construct into the TOM complex. Thus, the mitochondrial-targeting and TOM integration signal could be discriminated. A yeast two-hybrid assay revealed that the import sequence interacted with two intramolecular elements, the TMD and C-tail signal, and that it also interacted with the import receptor Tom20. Integral membrane proteins of the mitochondrial outer membrane are synthesized by cytoplasmic ribosomes as mature precursors and post-translationally integrated into the membrane (1Shore G.C. Mc Bride H.M. Millar D.G. Steenaart N.A.Y. Nguiyen M. Eur. J. Biochem. 1995; 227: 9-18Crossref PubMed Scopus (84) Google Scholar, 2Mihara K. BioEssays. 2000; 22: 364-371Crossref PubMed Scopus (43) Google Scholar). Unlike the matrix-targeted preproteins with cleavable presequences, the signals that target the outer membrane proteins are contained within the mature protein sequence. The import receptors of the preprotein translocase of the mitochondrial outer membrane (TOM 1The abbreviations used are: TOM, translocase of outer membrane; rTOM, rat TOM; AD, activating domain; BD, binding domain; BN-PAGE, blue native PAGE; ER, endoplasmic reticulum; GFP, green fluorescent protein; OMV, outer membrane vesicle(s); PBS, phosphate-buffered saline; TMD, transmembrane domain; bis-Tris, bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane. 1The abbreviations used are: TOM, translocase of outer membrane; rTOM, rat TOM; AD, activating domain; BD, binding domain; BN-PAGE, blue native PAGE; ER, endoplasmic reticulum; GFP, green fluorescent protein; OMV, outer membrane vesicle(s); PBS, phosphate-buffered saline; TMD, transmembrane domain; bis-Tris, bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane. complex (3Pfanner N. Geissler A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 339-349Crossref PubMed Scopus (415) Google Scholar), Tom70 (4Hurt E.C. Müller U. Schatz G. 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Chem. 1998; 273: 11527-11532Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). segment is a and is in and and it is to as the The of the internal import the signal is by the TOM and the by which the import signal and TMD the membrane membrane sequence of and to Tom22 S. and N. K. H. M. M. R. H. S. T. Sakaguchi M. T. Y. Mihara K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. N. K. M. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). the sequence is proteins a structural in the of of acidic residues the cytoplasmic domain the and in the The of acidic residues the intermembrane is The C-terminal of N. and S. Tom22 an with a of a C-terminal with a These to the signal of rat (rTOM22) that mitochondrial targeting and integration into the TOM complex using in and in rTOM22 is of residues and an sequence of to and were as K. H. M. M. R. H. S. T. Sakaguchi M. T. Y. Mihara K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Rat and the mitochondrial outer membrane were as N. T. R. Iwahashi J. Sakaguchi M. T. Mihara K. EMBO J. PubMed Scopus Google Scholar, H. A. Biochem. Google Scholar). constructs were by and the were into and used for the experiments in HeLa cells. Thus, of the constructs were the of the in for the constructs in which the three internal of rTOM22 were or in with the of green fluorescent protein the rTOM22 by or were with into the of the used in is Cell and cells were in with in an of was to the using was to HeLa and the cells were in for cells were cultured in in with for an of in was as and the cells were for with HeLa cells cultured for were in the of for 30 The cells were cultured and with phosphate-buffered with for 30 with PBS, and with for with and in PBS, the cells were for with or in with PBS, and using rTOM22 or were and analyzed using a confocal 2000; HeLa cells were cultured in for and with The cells were with the and N. Mihara K. J. Biochem. 1998; PubMed Scopus Google Scholar) and in of The cells were by through a and for The was for to the The was for 30 to separate the and the The were to and using The were by and the were analyzed by of Rat mitochondrial outer membrane were as by and H. A. Biochem. Google Scholar). The N. T. R. Iwahashi J. Sakaguchi M. T. Mihara K. EMBO J. PubMed Scopus Google Scholar) were in and for and with a for The was in and and and for The was and with and and for The were in the of the the rTOM22 to internal import sequence in the cytoplasmic TMD, C-tail signal, or was to the of the and the proteins were synthesized and with using a were in of of OMV, and 30 for The was with of the of of and in and for The OMV, which to the of the was and to by native was as H. Y. T. K. S. A. Mihara K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The HeLa cells the rTOM22 were in 30 of and was by for The was with of and through The were to using of rTOM22 into in of rTOM22 into was as S. Iwahashi J. Kida Y. Sakaguchi M. Mihara K. J. Cell Biol. 2000; 151: 277-288Crossref PubMed Scopus (165) Google Scholar). the of and of constructs was in of 30 for The were by for with and to or The were analyzed by using a two-hybrid were using the to the The of rTOM22 and three internal sequence of rTOM22 (the import sequence TMD and C-tail signal were by and the were downstream of the domain the or into the domain the were using the of two into S. was to the The were for to and The with an in the for of the mitochondrial targeting signal of rTOM22 (the sequence in 10-residue were introduced into rTOM22 and the and and in HeLa and intracellular localization was examined by confocal microscopy and cell fractionation. of and are in The mutant with a in the cytoplasmic to the which was also by cell The construct of residues also to the A and the constructs were to are for and and are for the constructs that mitochondrial localization in cytoplasmic segment and the TMD are for mitochondrial targeting of were introduced into rTOM22 the and in HeLa cells. The constructs were and were by and fluorescent were with a confocal fluorescent are rTOM22 constructs were in HeLa and the cells were into and the as The were to by using for constructs or the and were used as for and assembly of the rTOM22 into the TOM complex as revealed by were HeLa cells the constructs and were by The was into was to whereas the was to and the were analyzed by using We examined the constructs to the were into the TOM complex using The constructs with residues were integrated into the TOM complex H. Y. T. K. S. A. Mihara K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) The constructs with in the C-terminal intermembrane segment were integrated into the TOM complex with and were that the of the C-terminal segment affected assembly into the TOM complex. The signals were in the TOM complex or in the of the of the constructs with residues in the microscopy and cell that constructs were to as the proteins were in the by and for We for structure that residues an acidic α-helical in which residues were of the and residues the the segment in the domain as was contained in We the of the structure as as the acidic or residues in an of residues were in the middle portion of segment to the of residues in the of the constructs for the failed to mitochondrial were to the that residues be in the with the cell in residues in segment were by a the constructs and in were also to the These that the α-helical structure of segment is essential for mitochondrial We the of residues in three residues in segment were by in two residues were by the mitochondrial targeting was and the of of the in the the residues are residues and are the of the examined the of residues by with in A to in but the of the two with a These that residues in structure are for the targeting that the segment residues is essential for targeting and an acidic α-helical structure to the of rat and N. Tom22 were segment the N-terminal to the of N. Tom22 N. Nargang F.E. Baardman R. Neupert W. Lill R. Court D.A. J. Biol. Chem. 1998; 273: 11527-11532Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), the sequence was We segment the to that of N. A in the C-terminal of rTOM22 the of rTOM22 revealed that into mitochondrial targeting and in of the cells revealed that the constructs were to the with the in the in for that the C-terminal segment affected targeting efficiency. The construct with a of residues the segment residues was for the constructs and were to A and and These that of the segment or in residues was for the targeting of the The segment residues is in and residues and a structure We examined and residues were for the targeting efficiency. The mutant in which and residues in segment were to was to residues were for the and residues were to the construct was to the A and that residues are for the of intermembrane The TMD of rTOM22 for into the TOM examined the of the TMD residues in the TMD were by or residues the the constructs to the the with the A and Thus, a TMD with appropriate is essential for mitochondrial The of a in the TMD of Tom22 was in yeast R. B. K. Beilharz T. Lithgow T. FEBS Lett. PubMed Scopus Google Scholar), and was also in was with in the mutant was to the A and We examined the TMD of membrane proteins is for targeting or assembly into the TOM complex. the TMD of rTOM22 was by that of in HeLa the construct was to A and BN-PAGE, revealed that it failed to be integrated into the TOM complex that the import sequence in with the TMD of membrane proteins with an appropriate functioned as the targeting integration of rTOM22 into the TOM complex, of the TMD and the import sequence was A sequence in the TMD was for the Thus, the signals for mitochondrial targeting and for integration into the TOM complex could be The by the the components of the TOM complex of the three sequence in rTOM22 that are for mitochondrial targeting and TOM The three were or in to the of and binding of the constructs to the was by The construct the import sequence to the the constructs the import sequence and TMD and to the to a binding was was These that the import sequence in with TMD functioned as the signal, which was by the import receptors of the mitochondrial We the import sequence in the domain interacted with the TMD or C-tail signal using a yeast two-hybrid The import sequence to the domain interacted with the TMD to the domain was also an of the import sequence with the C-terminal signal whereas was TMD and the C-tail signal in These of three in membrane targeting and is that the import sequence with the TMD to as an import signal with a We examined the import sequence interacted with the cytoplasmic segment of the import receptors or Tom22, using a yeast two-hybrid interacted with and a as as of Tom20 and Tom22 T. T. K. Gratzer S. Schatz G. U. S. A. PubMed Scopus Google Scholar, A. Nargang F.E. Neupert W. Lill R. EMBO J. 1995; PubMed Scopus Google Scholar). The import sequence to interacted with both the cytoplasmic segment of Tom20 and Tom22, which were to and that the import signal was by Tom20 and Tom22 in the import is with the of the import by The rTOM22 into the import of N. Tom22 both Tom70 and and of the import (8Keil P. Pfanner N. FEBS Lett. 1993; 321: 197-200Crossref PubMed Scopus (62) Google Scholar, 10Rodriguez-Cousino N. Nargang F.E. Baardman R. Neupert W. Lill R. Court D.A. J. Biol. Chem. 1998; 273: 11527-11532Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, D.A. Nargang F.E. H. Neupert W. Lill R. Mol. Cell. Biol. 1996; PubMed Scopus (65) Google Scholar). with a of the C-terminal intermembrane domain are through a import N. Nargang F.E. Baardman R. Neupert W. Lill R. Court D.A. J. Biol. Chem. 1998; 273: 11527-11532Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, D.A. Nargang F.E. H. Neupert W. Lill R. Mol. Cell. Biol. 1996; PubMed Scopus (65) Google Scholar). the sequence of mammalian to N. Tom22 is as as examined the import of rTOM22 the of receptors as N. rTOM22 an Nout-Cin orientation in the outer and a C-terminal is by of K. H. M. M. R. H. S. T. Sakaguchi M. T. Y. Mihara K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We membrane integration of rTOM22 using the as a synthesized was integrated into the and the The import was by of the a of rTOM22 was the A and import was by but by A and Thus, import of rTOM22 the N. Tom22 is into the examined in rTOM22 by assembly of the rTOM22 constructs into the TOM complex revealed that rTOM22 was integrated into the complex, and of the integration and was into the but with the of of the of rTOM22 the in that the C-terminal segment affected import efficiency. to N. Tom22, a of was into and The in by and a to the C-terminal rTOM22 as by of the in and of rTOM22 N-terminal and C-terminal We that the of the C-terminal rTOM22 was into the through a a was the Thus, the C-tail segment of rTOM22 the import in a mammalian mitochondrial Tom22 is anchored to the outer membrane by an internal TMD in the Nout-Cin orientation. to the mitochondrial outer membrane proteins that are anchored to the membrane through the or C-terminal signal anchor rTOM22 three internal for targeting and integration into the TOM the TMD, a segment in the cytoplasmic domain the TMD (the import sequence), and the C-tail The of the in the cytoplasmic and intermembrane for N. Tom22, the and functions are of mammalian N. Tom22, the cytoplasmic segment residues functions as an essential internal import segment matrix-targeted in that it is in a and is the segment that functions as the internal import sequence of rTOM22 is to a 10-residue in the cytoplasmic and is for the of signal is the segment with an appropriate TMD as the mitochondrial targeting and membrane anchor are the components that structural of rTOM22 the import receptor Tom20 A yeast two-hybrid assay revealed that the import sequence interacted with the cytoplasmic of and N. a Tom22 mutant the C-terminal intermembrane domain is into in a D.A. Nargang F.E. H. Neupert W. Lill R. Mol. Cell. Biol. 1996; PubMed Scopus (65) Google Scholar), whereas the import is the import sequence in the cytoplasmic domain is N. Nargang F.E. Baardman R. Neupert W. Lill R. Court D.A. J. Biol. Chem. 1998; 273: 11527-11532Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), that the import sequence functions in the of experiments revealed in to N. Tom22, a of was into that the C-terminal rTOM22 was also into through the import These that the import sequence is in the import The yeast two-hybrid assay and experiments that the import sequence could be by sequence an acidic the be into the of Y. T. S. Mihara K. H. S. T. D. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) through an residues of the and the of be in that the both the import sequence and TMD and to the OMV, the import whereas the construct the import sequence These an of the TMD for the the signal by of the import sequence and The that the acidic residues the of the to be revealed that the rTOM22 mutant TMD was by the TMD was and integrated into the mitochondrial membrane but failed to be integrated into the TOM complex. These that the rTOM22 TMD is for assembly into the TOM complex. for S. Tom22 R. B. K. Beilharz T. Lithgow T. FEBS Lett. PubMed Scopus Google Scholar), a in the TMD is for the S. Tom22 the intermembrane domain is into the TOM complex S. Ryan M.T. K. C. J. Dekker P.J.T. Moczko M. Wagner R. M. B. A. Pfanner N. Nature. 1999; PubMed Scopus Google Scholar). both were by the TOM complex was the of Tom22, the the TOM complex to a core complex. the TMD of Tom22 is to the of the TOM complex S. Ryan M.T. K. C. J. Dekker P.J.T. Moczko M. Wagner R. M. B. A. Pfanner N. Nature. 1999; PubMed Scopus Google Scholar). with the that the rTOM22 TMD is in both integration into the TOM complex and of the assembly of the TOM complex, but the cytoplasmic segment is in is the orientation of membrane proteins the outer mitochondrial the and the the of the membrane is an J. Mol. Biol. 1992; PubMed Scopus Google Scholar, H. U. S. A. 1989; PubMed Scopus Google Scholar) N. Nargang F.E. Baardman R. Neupert W. Lill R. Court D.A. J. Biol. Chem. 1998; 273: 11527-11532Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), for N. Tom22 that orientation is by the the We also for TMD is by three the cytoplasmic and by the intermembrane and of the the rTOM22 mutant two the and two the intermembrane was to the mitochondrial outer membrane in the Nout-Cin orientation. H. M. and K. a protein in which the signal anchor of Tom70 was to that the upstream segment of the signal anchor was by the signal, Li and Shore (1Shore G.C. Mc Bride H.M. Millar D.G. Steenaart N.A.Y. Nguiyen M. Eur. J. Biochem. 1995; 227: 9-18Crossref PubMed Scopus (84) Google Scholar, Shore G. Science. 1992; PubMed Scopus Google Scholar) that the construct was into the outer membrane in the that of the of the signal anchor the cytoplasmic of the outer membrane is an for the Nout-Cin orientation. that the import sequence of rTOM22 interacted with the cytoplasmic domain of the import receptor it as the is that the three in rTOM22 were by the mitochondrial import of the import sequence downstream of TMD the orientation. of the was in for a the protein failed to be into the H. M. and K. These the of an the import sequence and the TMD the import the import sequence with the TMD to a and structure is by the import the of the segment the as in the of the signal anchor of S. Tom70 Shore G. 1996; PubMed Scopus Google Scholar). experiments revealed that the C-tail signal affected import efficiency. mammalian the C-terminal rTOM22 was into in the import a of the rTOM22 was into is in to the for N. Tom22 N. Nargang F.E. Baardman R. Neupert W. Lill R. Court D.A. J. Biol. Chem. 1998; 273: 11527-11532Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, D.A. Nargang F.E. H. Neupert W. Lill R. Mol. Cell. Biol. 1996; PubMed Scopus (65) Google Scholar). the yeast two-hybrid assay revealed that segment interacted with the import sequence in the cytoplasmic segment the of the import or the C-terminal signal with the components in the intermembrane A. P. B. A. Pfanner N. W. C. EMBO J. 22: PubMed Scopus Google Scholar) to membrane of is the in the the
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